Search tips
Search criteria 


Logo of annbotAboutAuthor GuidelinesEditorial BoardAnnals of Botany
Ann Bot. 2009 July; 104(1): 179–188.
Published online 2009 May 15. doi:  10.1093/aob/mcp108
PMCID: PMC2706734

Water-use responses of ‘living fossil’ conifers to CO2 enrichment in a simulated Cretaceous polar environment


Background and Aims

During the Mesozoic, the polar regions supported coniferous forests that experienced warm climates, a CO2-rich atmosphere and extreme seasonal variations in daylight. How the interaction between the last two factors might have influenced water use of these conifers was investigated. An experimental approach was used to test the following hypotheses: (1) the expected beneficial effects of elevated [CO2] on water-use efficiency (WUE) are reduced or lost during the 24-h light of the high-latitude summer; and (2) elevated [CO2] reduces plant water use over the growing season.


Measurements of leaf and whole-plant gas exchange, and leaf-stable carbon isotope composition were made on one evergreen (Sequoia sempervirens) and two deciduous (Metasequoia glyptostroboides and Taxodium distichum) ‘living fossil’ coniferous species after 3 years' growth in controlled-environment simulated Cretaceous Arctic (69°N) conditions at either ambient (400 µmol mol−1) or elevated (800 µmol mol−1) [CO2].

Key Results

Stimulation of whole-plant WUE (WUEP) by CO2 enrichment was maintained over the growing season for the three studied species but this pattern was not reflected in patterns of WUE inferred from leaf-scale gas exchange measurements (iWUEL) and δ13C of foliage (tWUEL). This response was driven largely by increased rates of carbon uptake, because there was no overall CO2 effect on daily whole-plant transpiration or whole-plant water loss integrated over the study period. Seasonal patterns of tWUEL differed from those measured for iWUEL. The results suggest caution against over simplistic interpretations of WUEP based on leaf isotopic composition.


The data suggest that the efficiency of whole-tree water use may be improved by CO2 enrichment in a simulated high-latitude environment, but that transpiration is relatively insensitive to atmospheric CO2 in the living fossil species investigated.

Key words: Water-use efficiency, elevated CO2, living fossil plants, conifers, paleoecology, ancient polar forests, stable carbon isotopes, stomatal conductance, canopy transpiration


The plant fossil record provides evidence for coniferous forests extending into the polar regions during the Mesozoic (251–65 Myr ago; Axelrod, 1984; Creber and Chaloner, 1985; Spicer and Chapman, 1990), a time when the global atmospheric CO2 concentration ([CO2]) was at least double that of the present-day, and the Earth was in a ‘greenhouse’ climate mode (Royer, 2006). These extinct high-latitude forests therefore experienced an environment unlike any on Earth today, characterized by a CO2-rich atmosphere, extreme seasonal variations in daylight, and winter temperatures above freezing (Tarduno et al., 1998). Although many studies have identified the direct and indirect action of CO2 enrichment on tree physiology (Medlyn et al., 1999, 2001; Ainsworth and Long, 2005; Ainsworth and Rogers, 2007), few have addressed the interaction between high [CO2] and a high-latitude light regime representative of these forests.

In the continuous light regime experienced by high-latitude paleoforests during summer, optimum growth would have required continuous gas exchange without excessive risk of catastrophic xylem embolism (Sperry et al., 1993). Extant conifers in present temperate and boreal biomes support part of the high transpirational demand during the day with water stored in stem sapwood, with losses being replenished during night when transpiration is low (Pallardy et al., 1995). However, in the continuous light of an ancient high-latitude summer, the value of a water-relations strategy employing stem capacitance and overnight recharging would be lost, placing a greater adaptive value on higher water-use efficiency (WUE), i.e. the ratio of CO2-fixation to water-loss (Jagels and Day, 2004).

Because increases in atmospheric CO2 concentration generally stimulate leaf photosynthesis (AL) and lower stomatal conductance to water vapour (gs) and transpiration rate (EL; Ainsworth and Rogers, 2007), we hypothesize that a CO2-rich atmosphere during the Mesozoic helped coniferous species in the polar regions to maintain a high instantaneous leaf WUE (iWUEL) throughout the growing period. Stimulation of WUE in these ancient CO2-rich atmosphere might also have offered the potential for greater tree height (Osborne and Beerling, 2002). However, experimental evidence indicates that the responses of AL and gs may be moderated by the extreme variations in daylight characteristics of high-latitude environments. Osborne and Beerling (2003) reported that a strong acclimation in photosynthetic capacity completely offset the CO2-stimulation of AL in three ‘living fossil’ coniferous species after several weeks of continuous illumination during a simulated ancient high-latitude summer (69°N). Moreover, conifers generally show weaker effects of elevated [CO2] on gs and EL than deciduous and evergreen broadleaf species (Saxe et al., 1998; Medlyn et al., 2001; Ainsworth and Long, 2005). Consequently, the expected beneficial effect of elevated [CO2] on WUE might have been reduced or lost during the summer in coniferous species growing in ancient high-latitude environments, but this proposition remains to be tested.

Leaf WUE can be assessed through instantaneous leaf gas-exchange measurements, and from leaf carbon isotope measurements (δ13C; Farquhar et al., 1982) which integrates the balance between carbon dioxide and water fluxes over long periods (for a review, see Dawson et al., 2002). To distinguish variations in the δ13C of source CO2 from the effects of metabolic processes, the δ13C signatures of organic material are translated to photosynthetic 13C discrimination (Δ13C; Farquhar et al., 1982; Farquhar and Richards, 1984). The model of Farquhar et al. (1982) relates Δ13C linearly to ci/ca, the ratio of intercellular (ci) to atmospheric (ca) CO2 mole fractions, which reflect leaf WUE integrated over the lifetime of the leaf. In the present study, both gas exchange and isotopic measurements are used to estimate leaf-scale WUE, and they are compared with WUE values derived from whole-plant gas exchange measurements to investigate the relationship between leaf-scale WUE and overall plant water use.

Previous studies indicated that large reductions in leaf-scale stomatal conductance and/or WUE at elevated [CO2] do not necessarily translate to reductions in rates of whole-tree transpiration (Wullschleger and Norby, 2001; Wullschleger et al., 2002). Explanations for this observation include: energy balance considerations, whereby increased canopy temperatures compensate for lower gs at elevated [CO2]; CO2-induced reductions in gs being limited to upper canopy leaves; and increases in leaf area for plants grown at elevated compared with ambient [CO2] that offset the reductions in water use due to partial stomatal closure (Field et al., 1995; Wilson et al., 1999; Wullschleger and Norby, 2001). However, we would not expect this last effect would be important in ancient paleoforests, since Royer et al. (2005) reported that, under simulated high-latitude conditions (69°N), elevated [CO2] decreased canopy leaf area in two out of five ‘living fossil’ tree species, while there were no statistically distinguishable changes in the others. Therefore, we might hypothesize that, in a warm high-latitude environment, decreases in gs would be translated into reductions in whole-plant transpiration (EP) over the growing season.

Here, measurements are reported of leaf- and whole-plant gas exchange, and leaf δ13C, made at bimonthly intervals throughout the growing season after 3 years' exposure of saplings of three ‘living fossil’ coniferous species to either current ambient (400 µmol mol−1) or elevated (800 µmol mol−1) [CO2] in controlled-environment simulated Cretaceous high-latitude conditions (69°N). The three studied species (Sequoia sempervirens, Metasequoia glyptostroboides and Taxodium distichum) belong to three genera known to occur in Cretaceous and Paleogene Arctic forests (Miller, 1977; Schweitzer, 1980; Aulenback and LePage, 1998; Yang and Jin, 2000). The use of ‘living fossil’ species, or extant taxa belonging to lineages characterized by little or no phenotypic change since the Mesozoic, as modern analogues for their congeneric ancestors provides a system to explore the ecophysiology of high-latitude paleoforests (Beerling and Osborne, 2002; Osborne and Beerling, 2003; Royer et al., 2003, 2005; Jagels and Day, 2004; Vann et al., 2004; Equiza et al., 2005; Jagels and Equiza, 2005; Llorens et al., 2009).

In the present study, the following hypotheses were tested: (1) atmospheric CO2 enrichment improves WUE of coniferous species growing in a simulated ancient high-latitude environment only in spring and autumn because, during summer, gs and transpiration rate are insufficiently reduced by elevated [CO2] to compensate for the acclimation of photosynthesis; and (2) despite the relatively low CO2 sensitivity of gs, a doubling of atmospheric CO2 reduces whole-plant water loss over the growing season. These two hypotheses also provide a framework for evaluating: (a) inferences about leaf or plant water use made using leaf δ13C measurements, and (b) the validity of instantaneous leaf-scale measurements to assess overall plant water use.


Experimental design

A Cretaceous Arctic environment was simulated in eight replicated growth rooms located in Sheffield, UK (53°N). Four were maintained at the current ambient [CO2] (400 µmol mol−1), and four at elevated [CO2] (800 µmol mol−1), similar to estimates for the Cretaceous based on geochemical proxies (Royer et al., 2001). In all the growth rooms, a daytime photosynthetic photon flux density (PPFD) of 300–400 µmol m−2 s−1 was provided using water-cooled sodium lamps (Sunbeam Hydrostar; Avon Gro-Lite Systems, Bristol, UK), and the photoperiod was changed weekly to simulate 69°N. Temperature within the growth rooms was raised by 5 °C compared with the outside air, and maintained above a minimum of +5 °C in the winter, to match proxy-based estimates of palaeotemperature at high latitudes for the Cretaceous (Beerling and Osborne, 2002; Royer et al., 2003). Relative humidity within the growth rooms was maintained above 75 % by using an automated misting system. Plants were grown in a silica sand–vermiculite–peat medium (13:5:2), watered twice daily via an automated drip irrigator. Fertilizer was applied as Rorison's nutrient solution, with a gradual increase in strength from 10 % to 50 % during the experiment as the plants grew larger, with the aim of providing a non-limiting nutrient supply. Full experimental details are provided elsewhere (Beerling and Osborne, 2002; Osborne and Beerling, 2003; Royer et al., 2003, 2005).

All saplings were grown from seed at the Llangwm Arboretum (Usk, UK) and acclimated to Sheffield climate for 2 months before transfer to the growth rooms (Beerling and Osborne, 2002). Saplings were 1 year old at the start of the experiment and were grown for further 3 years first in 2-L pots and later in 6-L pots. The coastal redwood (Sequoia sempervirens [D. Don] Endl) is evergreen, whereas the others, the dawn redwood (Metasequoia glyptostroboides Hu and Cheng) and the swamp cypress (Taxodium distichum Rich), are deciduous. Every year, leaf growth started in April and continued until September in all species. The three species are all members of the family Cupressaceae and have long fossil records at the generic level (>65 Myr), with ancestors that formed Cretaceous and Paleogene Arctic forests (Miller, 1977; Schweitzer, 1980; Aulenback and LePage, 1998; Yang and Jin, 2000).

Leaf gas exchange

Instantaneous measurements of leaf CO2 and H2O exchange rates were made using an open gas-exchange system (CIRAS-1; PP Systems, Hitchin, Herts, UK) on recently expanded upper canopy leaves during the third growing season. The leaves were illuminated with a quartz halide source providing a PPFD of 600 µmol m−2 s−1, which saturates photosynthesis in these species (Osborne and Beerling, 2003). Cuvette [CO2] and leaf-to-air vapour pressure difference were set to match growth conditions. Leaf temperature was maintained at 25 °C with a feedback control system. Values of AL, EL and the internal-to-ambient CO2 concentration (ci/ca) were calculated following von Caemmerer and Farquhar (1981), with iWUEL defined as the ratio of AL to EL.

Measurements were taken in April (14 h light d−1), June (24 h light d−1), July (24 h light d−1) and September (12 h light d−1), on a single leaf from two plants per species in each of four replicated growth rooms per CO2 treatment. Values of the in vivo carboxylation capacity of Rubisco (Vc,max) were calculated using the gas exchange data for each date, as reported previously by Osborne and Beerling (2003). Here Vc,max values were used to explore their relationship with Δ13C. Leaf dark respiration rates (μmol m−2 s−1) were also measured in the same leaves after 10 min dark adaptation. Leaf photorespiration rates (μmol m−2 s−1) were, then, calculated after Farquhar et al. (1980).

Whole-plant transpiration and water-use efficiency

Whole-plant CO2 and H2O fluxes were quantified using custom-built enclosures attached to a differential infrared gas analyser (CIRAS-1; PP Systems), using the same time intervals and a subset of the plants used for leaf measurements (for full details, see Royer et al., 2003, 2005). The gas exchange chambers were set-up within one of the growth rooms, and subjected to the same light and temperature regime. Radiant heating from the lamps was offset through the use of circulating water jackets. The incoming air was humidified by bubbling through water, and [CO2] controlled via the injection of pure CO2 into a mixing volume. An airflow rate of 4 L min−1 resulted in a mean residence time of 3 min for the air in each chamber, which was stirred continuously with two fans.

One plant per species was sampled from each of three replicate growth rooms per CO2 treatment. Each plant was sealed inside one of the chambers for 24 h, and hourly measurements were then made over the subsequent 24-h period. The hourly fluxes were integrated to obtain daily whole-plant net carbon uptake (AP) and transpiration (EP; Royer et al., 2003, 2005), with whole-plant water-use efficiency (WUEP) being, then, calculated as the quotient between AP and EP. Plant water use over the growing season was approximated by integrating EP over the study period.

Stable carbon isotope analyses

Foliar δ13C values were determined on the same leaves that were used for gas exchange. Oven-dried leaves were ground to a homogenized fine powder and δ13C determinations made on a 20-mg sub-sample using an ANCA GSL preparation module coupled to a 20–20 stable isotope analyser (PDZ Europa, Cheshire, UK). Measurements on the same sample had a reproducibility of 0·5‰. The isotopic composition of the sample (δ13Cs) was calculated as the sample 13C/12C ratio relative to the PDB standard (‰), and converted to discrimination values (Δ13C) using equation of Farquhar et al. (1989):

equation image

where δ13Ca is the isotopic composition of the atmosphere, estimated using flask samples taken from each of the eight growth rooms over two 6-h time periods, together with atmospheric and tank CO2 end-members. Concurrent measurements of [CO2] were obtained using the gas-exchange system. The δ13Ca of flask samples was measured with the same mass spectrometer, and mean values used to construct a Keeling plot (Keeling, 1958; Yakir and Sternberg, 2000). This yielded the following regression, which was then used to estimate δ13Ca from growth room records of [CO2] on a monthly basis equivalent to the sampling of leaf tissue:

equation image

Farquhar et al. (1982) formalized the relationship between Δ13C and leaf gas exchange with the well-validated model:

equation image

where a and b are constants representing isotope fractionation by diffusion through the stomatal pores (4·4 ‰) and by the enzyme ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco; 29 ‰), and d summarizes the effects of discrimination by CO2 dissolution, liquid phase diffusion, and possible discriminations during respiration and photorespiration (O'Leary, 1993). Since the value of d has been typically considered small, it is usually excluded from (3) and, thus, Δ13C is estimated as:

equation image

tWUEL was calculated following Farquhar and Richards (1984):

equation image

where [CO2] is the CO2 concentration in each growth room, the factor 1·6 is the ratio of gaseous diffusivities of CO2 and water vapour in the air, ci/ca is obtained from Δ13C by re-arranging (4), and D is the mean leaf-to-air water vapour pressure difference for each growth chamber, estimated from temperature and relative humidity measurements following Buck (1981), by assuming that leaf temperature equals air temperature.

Statistical analyses

Effects of [CO2] and species were assessed throughout the study period (except for total plant water loss) with two-way repeated-measures analysis of variance (ANOVAR) using means for each sampling date and growth chamber. The effects of [CO2] and species on whole-plant transpiration integrated over the study period were tested with two-way analysis of variance (ANOVA). Significant differences between means, as well as significant interactions between factors, were identified by taking P ≤ 0·05 as the level of significance. Relationships between variables were also analysed by means of least-square linear regressions using means for each species, sampling date and CO2 treatment.


Leaf gas exchange

Overall, gs measured under standardized conditions was lower in trees grown at elevated [CO2] than those from the ambient treatment (Table 1 and Fig. 1A–C). The effect of elevated [CO2] was largest in T. distichum in June and July, when gs fell by nearly 50 % from 244 mmol m−2 s−1 to 113 mmol m−2 s−1 (Fig. 1C). Instantaneous values of ci/ca, derived from the leaf gas exchange measurements, showed a significant CO2 effect on the balance between AL and gs, with higher overall ci/ca values in elevated than ambient [CO2] (Table 1 and Fig. 1D–F). Values of ci/ca decreased as a linear function of gs for plants grown at ambient [CO2], but this relationship was not significant in the case of plants grown at elevated [CO2] (Fig. 2A). However, instantaneous ci/ca decreased as a linear function of photosynthesis (AL) for plants grown at either ambient or elevated [CO2] (Fig. 2B).

Fig. 1.
Seasonal changes in (A–C) leaf stomatal conductance (gs), and (D–F) the ratio ci/ca derived from leaf gas exchange measurements for S. sempervirens, M. glyptostroboides and T. distichum growing at ambient or elevated [CO2] in a simulated ...
Fig. 2.
Instantaneous measurements of ci/ca plotted against leaf (A) stomatal conductance and (B) photosynthesis for S. sempervirens (triangles), M. glyptostroboides (circles) and T. distichum (squares) at ambient (open symbols) and elevated (closed symbols) ...
Table 1.
Results of two-way repeated-measures analysis of variance

Elevated [CO2] significantly increased iWUEL under standardized conditions (Table 1). However, this effect was restricted to spring and autumn in S. sempervirens and M. glyptostroboides (Fig. 3A and B), while it persisted throughout the year in T. distichum (Fig. 3C).

Fig. 3.
Seasonal changes in (A–C) instantaneous leaf WUE (iWUEL), (D–F) whole-plant WUE (WUEP), and (G–I) time-integrated leaf WUE (tWUEL) for S. sempervirens, M. glyptostroboides and T. distichum growing at ambient or elevated [CO2] in ...

Estimated leaf photorespiration rates were significantly lower in elevated than ambient [CO2], although the magnitude of this effect was more or less pronounced depending on the species and the sampling month (Table 1).

Whole-plant gas exchange

Direct measurements of whole-plant gas exchange under growth conditions showed that CO2 enrichment did not significantly affect daily transpiration (EP; Table 1 and Fig. 4A–C). After integration of these measurements over the study period, no significant CO2 effect was found on overall plant water use during this period (Fig. 5).

Fig. 4.
Seasonal changes in daily whole-plant transpiration (EP) for (A) S. sempervirens, (B) M. glyptostroboides and (C) T. distichum growing at ambient or elevated [CO2] in a simulated Cretaceous warm-Arctic environment. Values plotted are means ± s.e.m. ...
Fig. 5.
Whole-plant transpiration integrated over the study period using measurements of EP (Fig. 4) for S. sempervirens, M. glyptostroboides and T. distichum at ambient or elevated [CO2] in a simulated Cretaceous warm-Arctic environment. Values plotted ...

Calculation of WUEP from whole-plant measurements of AP and EP under growth conditions (Fig. 3D–F) gave lower values to those estimated from leaf gas exchange measurements (Fig. 3A–C) and showed a statistically significant, but attenuated, stimulation by elevated [CO2] across all species (Table 1). The non-significant CO2 × sampling month interaction indicated that CO2 effects on WUEP did not change significantly over the growing season, in contrast to what was found for iWUEL.

Stable carbon isotope discrimination

Values of Δ13C were significantly higher in plants growing under elevated [CO2] than those in the ambient treatment, in all species, and throughout the growing season (Table 1 and Fig. 6A–C). There was a significant negative relationship between Δ13C and Vc,max at both ambient and elevated [CO2] (Fig. 7A). Variation in Δ13C was also significantly correlated with variation in iWUEL in both CO2 treatments (Fig. 7B), but not with variation in WUEP (P = 0·624 and P = 0·358 for plants grown at ambient and elevated [CO2], respectively). However, trends in tWUEL (Fig. 3G–I) differed substantially from those found for iWUEL (Fig. 3A–C). Values of tWUEL were greater in the elevated than ambient CO2 treatment in the two deciduous species in the spring and at the beginning of the summer, but this CO2 effect was lost as the growing season progressed (Fig. 3H, I).

Fig. 6.
Seasonal changes in leaf carbon isotope discrimination (Δ13C) calculated from δ13C values for (A) S. sempervirens, (B) M. glyptostroboides and (C) T. distichum growing at ambient or elevated [CO2] in a simulated Cretaceous warm-Arctic ...
Fig. 7.
Leaf carbon isotope discrimination (Δ13C) calculated from δ13C values plotted against (A) Rubisco carboxylation capacity (Vc,max) and (B) instantaneous leaf WUE (iWUEL) for S. sempervirens (triangles), M. glyptostroboides (circles) and ...

As predicted by eqn (3), Δ13C values were significantly correlated with instantaneous measurements of ci/ca (Fig. 8). The regression equations explained 48 % and 36 % of variation in Δ13C for ambient and elevated [CO2] plants, respectively. Overall, Δ13C at a given ci/ca value was greater in the elevated than ambient [CO2] treatment, which is consistent with an influence of [CO2] on the y-intercept (offset) of eqn (3). Leaf Δ13C values at ambient [CO2] were poorly predicted from instantaneous ci/ca when using d = 0 ‰ (either assuming b = 29 ‰ or b = 27 ‰) in eqn (3) (Fig. 8). By contrast, d = 4 ‰ (assuming b = 29 ‰) provided a much better fit between predicted Δ13C values from instantaneous ci/ca and observed Δ13C values at ambient [CO2].

Fig. 8.
Leaf carbon isotope discrimination, (Δ13C, from δ13C values) vs. instantaneous ci/ca ratio (from leaf gas exchange measurements) for S. sempervirens (triangles), M. glyptostroboides (circles) and T. distichum (squares) at ambient (open ...


Water-use efficiency and transpiration estimated from gas exchange measurements

Increases in iWUEL with CO2 enrichment were restricted to the beginning and end of the growing season for S. sempervirens and M. glyptostroboides (Fig. 3A, B), in agreement with our first hypothesis. As anticipated, these patterns tracked seasonal variations in AL for both species (Osborne and Beerling, 2003), rather than gs (Fig. 1A, B). By contrast, CO2 enhancement of iWUEL in T. distichum was maintained throughout the study period (Fig. 3C) because continuous reductions in gs during the polar summer (Fig. 1C) offset the effects of acclimation on AL. The observed loss of AL stimulation by a doubling of atmospheric [CO2] in these coniferous species during summer (Osborne and Beerling, 2003) differ markedly from the situation in field-grown conifers of temperate environments, where AL typically shows a significant, season-long stimulation of >40 % in response to a doubling of CO2, which persists over a number of years (Ellsworth, 1999; Medlyn et al., 1999; Crous and Ellsworth, 2004). However, the low CO2 sensitivity of gs relative to that of AL is consistent with previous field studies showing that most conifer species respond to CO2 enrichment with small or non-significant decreases in gs (Saxe et al., 1998; Ellsworth, 1999; Medlyn et al., 2001), in contrast to the general pattern for angiosperms of significant decreases in this parameter (Ainsworth and Rogers, 2007).

At the whole-plant scale, the present results failed to support hypothesis (1), because the stimulation of WUEP by CO2 enrichment did not change significantly between sampling dates (Table 1). Because respiration of non-photosynthetic organs throughout the day, and respiration of autotrophic tissues in the dark, affect plant carbon balance, and non-transpirational (cuticular and lenticellular) water losses influence plant water economy, WUEP is expected to be lower than iWUEL (Ripullone et al., 2004). Accordingly, lower values of WUEP compared with iWUEL were found over the study period, irrespective of species and CO2 treatment, with differences between WUEP and iWUEL being greater in spring and autumn, i.e. when plants had some hours of darkness (Fig. 3A–F).

Despite values of WUEP for the three coniferous species being significantly higher at elevated than ambient [CO2], the relative insensitivity of EP to the CO2 treatment (Table 1 and Fig. 4) suggest that this response was driven largely by increased canopy assimilation rates (Royer et al., 2005). The non-significant effect of CO2 enrichment on plant water loss over the study period (Fig. 5) is in accordance with previous studies showing that canopies may be substantially decoupled from the atmosphere, with transpiration from the canopy being relatively insensitive to changes in gs (Eamus, 1991, Wullschleger et al., 2002). Therefore hypothesis (2) is rejected as an important component of the physiological ecology of conifer species growing under CO2 enrichment in a warm, high-latitude light environment.

Nevertheless, the sustained improvement of WUEP over the growing season in response to a CO2-enriched atmosphere might have benefited the growth of coniferous species inhabiting ancient high-latitude environments, even during the continuous light period. WUEP is likely to be an important determinant of maximum tree height, because xylem water potential declines markedly with height, imposing a hydraulic constraint on stomata and therefore a CO2-diffusion limitation on photosynthesis (Koch et al., 2004). Stimulation of photosynthesis and WUE via enhanced CO2 offsets this hydraulic mechanism, offering the potential for greater maximum tree height in ancient CO2-rich atmospheres (Osborne and Beerling, 2002). Indeed, it has been reported that the productivity of high-latitude paleoforests was comparable with that of living temperate deciduous forests, with trees reaching diameters of 1 m and heights of up to 40 m (Francis, 1991; Greenwood and Basinger, 1994; Williams et al., 2003). Therefore, although a CO2-enriched atmosphere might have brought about no significant improvement in water economy of coniferous species growing in Cretaceous high-latitude regions, it might have helped trees to reach heights comparable to those found in present temperate deciduous forests.

Inference of water use from isotope data

Throughout the study period, but especially during the high-latitude summer, all three coniferous species showed higher foliage Δ13C values when grown under high [CO2] (Table 1 and Fig. 6). This result is in accordance with: (a) the observed reduction of carboxylation capacity in plants growing under CO2 enrichment during summer (Osborne and Beerling, 2003), and (b) the marginal effect of elevated [CO2] on the stomatal conductance of these species (Table 1 and Fig. 1A–C). A decrease in plant carboxylation capacity, in the absence of a significant change in gs, should lead to a rise in Δ13C (Farquhar et al., 1989) and, accordingly, a significant negative relationship was found between Δ13C and the maximum rate of RuBP carboxylation (Vc,max; Fig. 7A). The higher ci/ca ratios (Fig. 1D–F) and Δ13C values observed in plants growing under elevated [CO2] compared with controls are in agreement with some previous studies (Picon et al., 1996; Bryant et al., 1998), although chamber and FACE (free-air CO2 enrichment) studies have usually shown that the co-ordination of stomatal conductance and photosynthesis is not substantially altered by CO2 enrichment, with ci/ca being typically insensitive to atmospheric CO2 (Drake et al., 1997; Xu and Hsiao, 2004; Ainsworth and Long, 2005).

As predicted by theory (Farquhar et al., 1989), a significant negative relationship was found between Δ13C and iWUEL (Fig. 7B). However, plants grown at ambient [CO2] showed lower iWUEL, but also lower Δ13C values when compared with plants grown at elevated [CO2]. Therefore, the present data support previous studies showing that lower Δ13C may not always indicate higher iWUEL, or translate into higher WUE at the whole-plant level (Condon et al., 2004; Seibt et al., 2008).

Seasonal patterns of tWUEL also differed from those measured for iWUEL for the three species studied (Fig. 3). All of these discrepancies may be interpreted from different, but not mutually exclusive, perspectives. The first potential mechanism is a ‘smoothing’ effect in the isotopic composition of tree leaves, whereby the carbohydrate used to construct new foliage is a mixture of recently fixed and stored carbon pools (Keel et al., 2007). Secondly, discrepancies between tWUEL and iWUEL may have resulted from differences between D (leaf-to-air water vapour pressure difference) and PPFD averaged over the course of the experiment, as compared with values in the cuvette during instantaneous measurements (Xu and Hsiao, 2004; Cernusak et al., 2007). Thirdly, part of the discrepancy may relate to the fact that ci/ca is calculated differently from instantaneous gas exchange measurements than from isotopic measurements (Cernusak et al., 2007). Finally, discrepancies may arise from the fact that eqn (4) excludes the term d, which summarizes the fractionation caused by dissolution of CO2 and liquid phase diffusion, photorespiration and dark respiration (Farquhar et al., 1989). Previous studies have reported substantial differences between observed Δ13C and the predictions of eqn (4), with eqn (4) overestimating discrimination under ambient [CO2] conditions (von Caemmerer and Evans, 1991; Farquhar and Lloyd, 1993; Gillon and Griffiths, 1997; Lauteri et al., 1997; Seibt et al., 2008). Few direct estimates of d exist in the literature and, as far as we know, all of them are for species growing at current [CO2]: approx. 3 ‰ in wheat (Evans et al., 1986), near-zero for barley (Hubick and Farquhar, 1989), approx. 1 ‰ for peanut (Hubick, 1990), 4 ‰ for Ficus insipida (Cernusak et al., 2007) and 3 ‰ for several tropical species (Cernusak et al., 2008). In the present data, a value of d = 4 ‰ provided a much better fit (assuming b = 29 ‰) between predicted (from instantaneous ci/ca) and observed Δ13C values at ambient [CO2] than using d = 0 ‰ (either assuming b = 29 or 27 ‰; Fig. 8). An effect of [CO2] on d would be consistent with the suppression of photorespiration at elevated [CO2] inferred from the present data (Table 1), but this aspect of the results requires further investigation.


The data indicate that leaf- and whole-plant-scale WUE in conifers are significantly enhanced by elevated [CO2] under a simulated warm Cretaceous high-latitude environment. These responses are driven primarily by photosynthesis rather than stomatal conductance, and are highly seasonal and species-specific at the leaf but not the plant scale. Overall, the present data suggest that, while the efficiency of whole-tree water use may be improved by CO2 enrichment in this system, transpiration is relatively insensitive to atmospheric CO2.


We thank Steve Ellin for technical assistance and Dana Royer for advice and comments. This study was supported by a European Union Marie Curie postdoctoral fellowship to L.L. (contract no. EVK2-CT-2002-50022).


Abbreviations used

APDaily whole-plant net carbon uptake (mmol CO2 plant−1 d−1)
ALNet leaf photosynthetic rate (μmol CO2 m−2 s−1)
EPDaily whole-plant transpiration (mol H2O plant−1 d−1)
ELLeaf transpiration rate (mmol H2O m−2 s−1)
gs,Leaf stomatal conductance (mmol H2O m−2 s−1)
iWUELInstantaneous leaf water-use efficiency (mmol CO2 mol−1 H2O)
PPFDPhotosynthetic photon flux density (μmol m−2 s−1)
tWUELTime-integrated leaf water-use efficiency (mmol CO2 mol−1 H2O)
WUEPWhole-plant water-use efficiency (mmol CO2 mol−1 H2O)
Δ13CDiscrimination against 13C (‰)
δ13CStable carbon isotope composition (‰)
δ13Caδ13C of the atmosphere
δ13Csδ13C of the sample
Vc,maxRubisco carboxylation capacity (μmol CO2 m−2 s−1)


  • Ainsworth EA, Long SP. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytologist. 2005;165:351–372. [PubMed]
  • Ainsworth EA, Rogers A. The response of photosynthesis and stomatal conductance to rising [CO2]: mechanisms and environmental interactions. Plant, Cell & Environment. 2007;30:258–270. [PubMed]
  • Aulenback KR, LePage BA. Taxodium wallisii sp. nov.: first occurrence of Taxodium from the Upper Cretaceous. International Journal of Plant Sciences. 1998;159:367–390.
  • Axelrod DI. An interpretation of Cretaceous and Tertiary biota in polar regions. Palaeogeography, Palaeoclimatology, Palaeoecology. 1984;45:105–147.
  • Beerling DJ, Osborne CP. Physiological ecology of Mesozoic polar forests in a high CO2 environment. Annals of Botany. 2002;89:1–11. [PubMed]
  • Bryant J, Taylor G, Frehner M. Photosynthetic acclimation to elevated CO2 is modified by source:sink balance in three component species of chalk grassland swards grown in a free air carbon dioxide enrichment (FACE) experiment. Plant, Cell & Environment. 1998;21:159–168.
  • Buck AL. New equations for computing vapour pressure and enhancement factor. Journal of Applied Meteorology. 1981;20:1527–1532.
  • von Caemmerer S, Evans JR. Determination of the average partial pressure of CO2 in chloroplasts from leaves of several C3 plants. Australian Journal of Plant Physiology. 1991;18:287–305.
  • von Caemmerer S, Farquhar GD. Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta. 1981;153:376–387. [PubMed]
  • Cernusak LA, Winter K, Aranda J, Turner BL, Marshall JD. Transpiration efficiency of a tropical pioneer tree (Ficus insipida) in relation to soil fertility. Journal of Experimental Botany. 2007;58:3549–3566. [PubMed]
  • Cernusak LA, Winter K, Turner BL. Conifers, angiosperm trees, and lianas: growth, whole-plant water and nitrogen use efficiency, and stable isotope composition (δ13C and δ18O) of seedlings grown in a tropical environment. Plant Physiology. 2008;148:642–659. [PubMed]
  • Condon AG, Richards RA, Rebetzke GJ, Farquhar GD. Breeding for high water-use efficiency. Journal of Experimental Botany. 2004;55:2447–2460. [PubMed]
  • Creber GT, Chaloner WG. Tree growth in the Mesozoic and early Tertiary and the reconstruction of palaeoclimates. Palaeogeography, Palaeoclimatology, Palaeoecology. 1985;52:35–60.
  • Crous KY, Ellsworth DS. Canopy position affects photosynthetic adjustments to long-term elevated CO2 concentration (FACE) in aging needles in a mature Pinus taeda forest. Tree Physiology. 2004;24:961–970. [PubMed]
  • Dawson TE, Nambelli S, Plamboeck AH, Templer PH, Tu KP. Stable isotopes in plant ecology. Annual Review of Ecology and Systematics. 2002;33:507–559.
  • Drake BG, Gonzàlez-Meler MA, Long SP. More efficient plants: a consequence of rising atmospheric CO2? Annual Review of Plant Physiology and Plant Molecular Biology. 1997;48:609–639. [PubMed]
  • Eamus D. The interaction of rising CO2 and temperatures with water use efficiency. Plant, Cell & Environment. 1991;14:843–852.
  • Ellsworth DS. CO2 enrichment in a maturing pine forest: are CO2 exchange and water status in the canopy affected? Plant, Cell & Environment. 1999;22:461–472.
  • Equiza MA, Day ME, Jagels R. Physiological responses of three deciduous conifers (Metasequoia glyptostroboides, Taxodium distichum and Larix laricina) to continuous light: adaptive implications for the early Tertiary polar summer. Tree Physiology. 2005;26:353–364. [PubMed]
  • Evans JR, Sharkey TD, Berry JA, Farquhar GD. Carbon isotope discrimination measured concurrently with gas exchange to investigate CO2 diffusion in leaves of higher plants. Australian Journal of Plant Physiology. 1986;13:281–292.
  • Farquhar GD, Lloyd J. Carbon and oxygen isotope effects in the exchange of carbon dioxide between terrestrial plants and the atmosphere. In: Ehleringer JR, Hall AE, Farquhar GD, editors. Stable isotopes and plant carbon-water relations. San Diego, CA: Academic Press; 1993. pp. 47–70.
  • Farquhar GD, Richards RA. Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes. Australian Journal of Plant Physiology. 1984;11:539–552.
  • Farquhar GD, von Caemmerer S, Berry JA. A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta. 1980;149:78–90. [PubMed]
  • Farquhar GD, Ball MC, von Caemmerer S, Roksandic Z. Effect of salinity and humidity on δ13C value of halophytes – evidence for diffusional isotope fractionation determined by the ratio of intercellular/atmospheric partial pressure of CO2 under different environmental conditions. Oecologia. 1982;52:121–124.
  • Farquhar GD, Ehleringer JR, Hubick KT. Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology. 1989;40:503–537.
  • Field CB, Jackson RB, Mooney HA. Stomatal responses to increased CO2: implications from the plant to global scale. Plant, Cell & Environment. 1995;18:1214–1225.
  • Francis JE. The dynamics of polar fossil forests of Axel Heiberg island, Canadian Arctic Archipelago. Geological Survey of Canada Bulletin. 1991;403:29–38.
  • Gillon JS, Griffiths H. The influence of (photo)respiration on carbon isotope discrimination in plants. Plant, Cell & Environment. 1997;20:1217–1230.
  • Greenwood DR, Bassinger JF. The paleoecology of high-latitude Eocene swamp forests of Axel Heiberg island, Canadian Arctic Archipelago. Review of Paleobotany and Palynology. 1994;81:83–97.
  • Hubick KT. Effects of nitrogen source and water limitation on growth, transpiration efficiency and carbon-isotope discrimination in peanut cultivars. Australian Journal of Plant Physiology. 1990;17:413–430.
  • Hubick KT, Farquhar GD. Carbon isotope discrimination and the ratio of carbon gained to water lost in barley cultivars. Plant, Cell & Environment. 1989;12:795–804.
  • Jagels R, Day ME. The adaptive physiology of Metasequoia to Eocene high-latitude environments. In: Hemsley AR, Poole I, editors. The evolution of plant physiology: from whole plants to ecosystems. London: Elsevier; 2004. pp. 401–425.
  • Jagels R, Equiza MA. Competitive advantages of Metasequoia in warm high latitudes. In: LePage BA, Williams CJ, Yang H, editors. The geobiology and ecology of Metasequoia. Dordrecht: Springer-Verlag; 2005. pp. 335–349.
  • Keel SG, Siegwolf RTW, Jäggi M, Körner C. Rapid mixing between old and new C pools in the canopy of mature forest trees. Plant, Cell & Environment. 2007;30:963–972. [PubMed]
  • Keeling CD. The concentration and isotopic abundances of atmospheric carbon dioxide in rural areas. Geochimica et Cosmochimica Acta. 1958;13:322–334.
  • Koch GW, Sillett SC, Jennings GM, Davis SD. The limits to tree height. Nature. 2004;428:851–854. [PubMed]
  • Lauteri M, Scartazza A, Guido MC, Brugnoli E. Genetic variation in photosynthetic capacity, carbon isotope discrimination and mesophyll conductance in provenances of Castanea sativa adapted to different environments. Functional Ecology. 1997;11:675–683.
  • Llorens L, Llusià J, Murchie EH, Peñuelas J, Beerling DJ. Monoterpene emissions and photoinhibition of ‘living fossil’ trees grown under CO2 enrichment in a simulated Cretaceous polar environment. Journal of Geophysical Research. 2009;114:G01005. doi:10.1029/2008JG000802.
  • Medlyn BE, Badeck F-W, De Pury DGG, et al. Effects of elevated [CO2] on photosynthesis in European forest species: a meta-analysis of model parameters. Plant, Cell & Environment. 1999;22:1475–1495.
  • Medlyn BE, Barton CVM, Broadmeadow MSJ, et al. Stomatal conductance of forest species after long-term exposure to elevated CO2 concentration: a synthesis. New Phytologist. 2001;149:247–264.
  • Miller CN. Mesozoic conifers. The Botanical Review. 1977;43:217–280.
  • O'Leary MH. Biochemical basis of carbon isotope fractionation. In: Ehleringer JR, Hall AE, Farquhar GD, editors. Stable isotopes and plant carbon – water relations. New York, NY: Academic Press; 1993. pp. 19–28.
  • Osborne CP, Beerling DJ. Sensitivity of tree growth to a high CO2 environment: consequences for interpreting the characteristics of fossil woods from ancient ‘greenhouse’ worlds. Palaeogeography, Palaeoclimatology, Palaeoecology. 2002;182:15–29.
  • Osborne CP, Beerling DJ. The penalty of a long, hot summer: photosynthetic acclimation to high CO2 and continuous light in ‘living fossil’ conifers. Plant Physiology. 2003;133:803–812. [PubMed]
  • Pallardy SG, Cermak J, Ewers FW, Kaufmann MR, Parker WC, Sperry JS. Water transport dynamics in trees and stands. In: Smith WK, Hinckley TM, editors. Resource physiology of conifers. San Diego, CA: Academic Press; 1995. pp. 301–389.
  • Picon C, Guehl JM, Aussenac G. Growth dynamics, transpiration and water-use efficiency in Quercus robur plants submitted to elevated CO2 and drought. Annales des Sciences Forestieres. 1996;53:431–446.
  • Ripullone F, Lauteri M, Grassi G, Amato M, Borghetti M. Variation in nitrogen supply changes water-use efficiency of Pseudotsuga menziesii and Populus × euroamericana; a comparison of three approaches to determine water-use efficiency. Tree Physiology. 2004;24:671–679. [PubMed]
  • Royer DL. CO2-forced climate thresholds during the Phanerozoic. Geochimica et Cosmochimica Acta. 2006;70:5665–5675.
  • Royer DL, Berner RA, Beerling DJ. Phanerozoic atmospheric CO2 change: evaluating geochemical and paleobiological approaches. Earth-Science Reviews. 2001;54:349–392.
  • Royer DL, Osborne CP, Beerling DJ. Carbon loss by deciduous trees in a CO2-rich ancient polar environment. Nature. 2003;424:60–62. [PubMed]
  • Royer DL, Osborne CP, Beerling DJ. Contrasting seasonal patterns of carbon gain in evergreen and deciduous trees of ancient polar forests. Paleobiology. 2005;31:141–150.
  • Saxe H, Ellsworth DS, Heath J. Tree and forest functioning in an enriched CO2 atmosphere. New Phytologist. 1998;139:395–436.
  • Schweitzer HJ. Environment and climate in the early Tertiary of Spitsbergen. Palaeogeography, Palaeoclimatology, Palaeoecology. 1980;30:297–311.
  • Seibt U, Rajabi A, Griffiths H, Berry JA. Carbon isotopes and water use efficiency: sense and sensitivity. Oecologia. 2008;155:441–454. [PubMed]
  • Sperry JS, Alder NN, Eastlack SE. The effect of reduced hydraulic conductance on stomatal conductance and xylem cavitation. Journal of Experimental Botany. 1993;44:1075–1082.
  • Spicer RA, Chapman JL. Climate change and the evolution of high-latitude terrestrial vegetation and floras. Trends in Ecology and Evolution. 1990;5:279–284. [PubMed]
  • Tarduno JA, Brinkman DB, Renne PR, Cottrell RD, Scher H, Castillo P. Evidence for extreme climatic warmth from Late Cretaceous arctic vertebrates. Science. 1998;282:2241–2244. [PubMed]
  • Vann DR, Williams CJ, LePage BA. Experimental evaluation of photosystem parameters and their role in the evolution of stand structure and deciduousness in response to palaeoclimate seasonality in Metasequoia glyptostroboides (Hu et Cheng) In: Hemsley AR, Poole I, editors. The evolution of plant physiology: from whole plants to ecosystems. London: Elsevier; 2004. pp. 427–445.
  • Williams CJ, Johnson AH, LePage BA, Vann DR, Sweda T. Reconstruction of Tertiary Metasequoia forests. II. Structure, biomass, and productivity of Eocene floodplain forests in the Canadian Arctic. Paleobiology. 2003;29:271–292.
  • Wilson KB, Carlson TN, Bunce JA. Feedback significantly influences the simulated effect of CO2 on seasonal evapotranspiration from two agricultural species. Global Change Biology. 1999;5:903–917.
  • Wullschleger SD, Norby RJ. Sap velocity and canopy transpiration in a sweetgum stand exposed to free-air CO2 enrichment (FACE) New Phytologist. 2001;150:489–498.
  • Wullschleger SD, Gunderson CA, Hanson PJ, Wilson KB, Norby RJ. Sensitivity of stomatal and canopy conductance to elevated CO2 concentration – interacting variables and perspectives of scale. New Phytologist. 2002;153:485–496.
  • Xu L-K, Hsiao TC. Predicted versus measured photosynthetic water-use efficiency of crop stands under dynamically changing field environments. Journal of Experimental Botany. 2004;55:2395–2411. [PubMed]
  • Yakir D, Sternberg LS. The use of stable isotopes to study ecosystem gas exchange. Oecologia. 2000;123:297–311.
  • Yang H, Jin J. Phytogeographic history and evolutionary stasis of Metasequoia: geologic and genetic information contrasted. Acta Paleontologica Sinica. 2000;39:288–307.

Articles from Annals of Botany are provided here courtesy of Oxford University Press